Separation of Sugar Acetates by Chromatography1 - Journal of the

J. Am. Chem. Soc. , 1945, 67 (4), pp 527–529. DOI: 10.1021/ja01220a009. Publication Date: April 1945. ACS Legacy Archive. Note: In lieu of an abstra...
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April, 1945

SEPARATION OF SUGAR ACETATESBY CIIROMATOGRAPIIY

kin’s nomenclature, based on X-ray diffraction patterns, seems the most promising available to correlate properly the polymorphic forms of a number of different triglycerides and to associate them with corresponding forms of other long chain compounds. Acknowledgment.-For advice and encouragement during the course of this work the writer expresses his gratitude t o A. S. Richardson and R. H. Ferguson of this Laboratory.

Summary The “even” members of the homologous series of triglycerides, trilaurin through tristearin, have been studied by X-ray diffraction and thermal methods and found to exhibit monotropic trimorphism. This is in line with the conclusions of previous observers, notably Malkin. Manifestation of different crystal structures by a given triglyceride was clearly established by the classical work of Malkin, et al., as the underlying cause of the multiple melting of triglycerides. The names of polymorphic forms were based by

[CONTRIBUTION FROM

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Malkin upon X-ray diffraction patterns in a manner to relate logically the triglyceride forms to corresponding forms of other long chain compounds. I t resulted that the forms for tristearin, etc., were called (by Malkin) gamma (glassy), alpha, and beta in order of increasing melting point. Unfortunately, according to the present study, there appears to have been a faulty association of X-ray diffraction pattern with melting point in the work of Malkin. This association was correct in the case of the highest melting beta form. However, the lowest melting form (called gamma, glassy or vitreous by Malkin) actually exhibits Malkin’s alpha pattern and in accordance with Malkin’s original intention is therefore named the aleha form. The intermediate form (called alpha by Malkin) actually exhibits Malkin’s beta prime pattern (not reported by him for tristearin, etc., but for mixed glycerides). It is therefore named the beta prime form. The gamma name and pattern and the concept of the glassy state of triglycerides should be eliminated. IVORYDALE 17, OHIO

RECEIVED DECEMBER 4, 194 t

CHEMICAL LABORATORY OF THEOHIOSTATEUNIVERSITY ]

Separation of Sugar Acetates by Chromatography’ BY W. H. MCNEELY,~ W. W. BINKLEY~ AND M. L. WOLFROM Application of Chromatographic methods to the separation of sugars has been made by H a y a ~ h i ,who ~ reported the separation of Dglucose and sucrose on a charcoal column. Reich5 employed the colored p-phenylazobenzoates of the sugars and this general method has been extended successfully by Coleman and associates6 to a very considerable number of typical separations. Employing the procedure of fractional elution (liquid or flowing chromatogram), Talley, Reynolds and Evans’ and Jones8 have applied chromatographic methods to the separation of sugar derivatives. Bells has utilized the partition (chloroform-watersilica gel) chromato( 1 ) Presented before the Division of Sugar Chemistry and Technology at the 108th meeting of the American Chemical Society, New York, N. Y., September 15, 1944. (2) Hoffmann-La Roche Post-doctoral Fellow of The Ohio State University Research Foundation. (3) Sugar Research Foundation Post-doctoral Fellow of The Ohio State University Research Foundation. (4) F. Hayashi. J . Biochcm. (Japan), 16, 1 (1932); C. A , . I T , 8 (1933). (6) W. S. Reich, Compl. rend., 408, 589, 748 (1939); Biochcm. J . , an, 1000 (1939). (6) G. H. Coleman, A. G. Farnham and A. Miller, THISJOURNAL, $4, 1501 (1942); G. H . Coleman and C. M. McCloskey, ibid., 86, 1588 (1943); cf. J. K . Mertzweiller. D. M. Carney and F . F . Farley, ibid., 2367. (7) E. E. Talley, D . D. Reynolds and W. L. Evans, ibid.. 66, 573 (1943). (8) J. K. N. Jones, J . Chcm. SOC.,333 (1944). (9) D. J. Bell, ibid., 473 (1944).

graphic procedure of Martin and Synge‘O in the establishment of a new 0-tetramethyl-D-glucopyranose end-group assay for polysaccharides. The Molisch reagent was used by Bell to locate the zones on the developed, extruded and ovendried column. Although the above-mentioned chromatographic procedures represent a considerable advance over methods previously available for the separation of sugar mixtures, they have disadvantages. We wish to report herein the successful application of the chromatographic brush method established by Zechmeister and collaborators’ to the separation of the well-characterized sugar acetates. This method consists in extruding the developed, colorless Chromatogram and making the &zonesvisible by brushing a line along the length of the column with a brush dipped in a reagent capable of forming a colored reaction product with the adsorbed substance. Such a procedure avoids the previous necessity of either working with colored sugar derivatives or resorting to empirical chromatography. A search for a suit(10) A. J. P. Martin and R. L. M. Synge, Biochcm. J . , S I , 1358 (1941); A. H. Gordon, A . J. P. Martin and R . L . M. Synge, ibid., 87, 79, 86, 92 (1943). (11) L. Zechmeister. L. de Cbolnoky and (Mlle.) E. Ujhelyi, Bull. SOL. ckim. biol., 18, 1885 (1936); L. Zechmeister and 0. Frehden, ibid., 93, 458 (1940); L. Zechmeister and W. H. McNeely, THIS 64, 1919 (1942): L. Zechmeister, W. H. McNeely and JOURNAL, G. S6lyom, ibid., 1922.

W. H. MCNEELY,W. W. BINKLRY A N D M. I,. WOLFROM

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Vol. 67

moderate speed and leaving the trisaccharide acetates near the top of the column. Application of these results led to the separation of the acetates of the following two component sugar mixtures (1 : 1): P-D-glucose from keto-D-fructose, @-maltose,sucrose, and raffinose ; P-maltose from sucrose, and raffinose; raffinose from sucrose. The reagent was also suitable for the’detection of the fully acetylated sugar alcohols, and the separation of (2euo)-sorbitol hexaacetate from L-rhamnito1 pentaacetate was effected readily. The results of this investigation are tabulated in Table 11. The recovery of each component of the mixtures was nearly quantitative. From the properties of the recovered fractions recorded in Table SUGAR AND SUGAR ALCOHOL ACETATESON “MAGNBSOL” 11, it can be seen that the purity of each coxnponent was excellent without recrystallization. WITH ALKALINE PERMANGANATE AS BRUSH REAGENT In the examples shown in Table XI, the upper and Substance Time, sec. lower zones pf the chromatograms were separated kelo-D-Fructose pentaacetate 10 by relatively large interzones, enabling each com8-D-Glucopyranose pentaacetate 30 ponent to be recovered in a high state of purity. 60 @-hfaltoseoctaacetate Preliminary experiments indicated that the dif Sucrose octaacetate 120-180 ferences in adsorption affinity between a number of Raffinose hendecaacetate 120-180 pairs of substances such as a- and P-D-ghCOSe (levo)-Sorbitol hexaacetate 180-240 pentaacetate were smaller and that rechromatoL-Rhamnitol pentaacetate 240-300 graphing probably would be necessary for their Exploratory experiments demonstrated that the separation. sugar acetates fell into general groups determined The separation of the sugar acetates by applicaby small changes in the nature of the developing tion of the chromatographic brush method shown agent. A ratio of 500 parts by volume of benzene in the present work offers a number of advantages to 1 part of ethanol moved the fully acetylated for the isolation, separation and identification of monosaccharides tested, except keto-D-fructose sugars over the methods previously available. pentaacetate, down the column while the acetates The empirical liquid chromatogram or fractional of the di- and trisaccharides were held near the elution method is tedious and is very difficult to top. A 100 to 1 ratio readily washed the mono- apply to the problem of isolating all of the comsaccharide acetates off the column while moving ponents of a complex, naturally occurring, mixture the disaccharide acetates to the bottom with a of sugars. While the use of colored sugar derivaable brush reagent, capable of locating the zones of the invisible chromatogram, led to the selection of aqueous alkaline permanganate. This reagent was suitable for non-reducing as well as for reducing sugars, and even for sugar alcohols. The brush reagent streak on the extruded column reacted with the invisible zones a t rates characteristic of the particular sugar acetate constituting the zone (Table I). Maxiy of the common adsorbents were found to be suitable in some degree but “Magnesol,” a synthetic hydrated magnesium acid silicate (211igO.5SiO2), was chosen as the most versatile. TABLE I COMPARATIVE RATESOF DISTINCT Z o m FORMATION FOR

TABLE I1 CHROMATOGRAPHIC SEPARATION OF SUGAR ACETATEAXD SUGAR ALCOHOL ACETATEPAIRSON “MAGNESOL” (50 g.“MAGNESOL”-“CELITE”MIXTURE5 : 1 )

Substance

Wt., mg.

Developing agent Vol. ratio, Relative benzeneVol., zone ethanol cc. location

8-D-Glucose pentaacetate 124’0 2 W : l 8-Maltose octaacetate 130.0 B-D-Glucose pentaacetate 125’1 200:l Sucrose octaacetate 125.6 8-D-Glucose pentaacetate 126’5 2OO:l Raffinose hendecaacetate 123.9 8-D-Glucose pentaacetate 125’2 500:l keto-D-Fructose pentaacetate 127.7 @-Maltoseoctaacetate 125’2 300:2.25 Sucrose octaacetate 126.8 @-Maltoseoct aacet ate 124’7 1 W : l Raffinose hendecaacetate 125.1 Sucrose octaacetate 125.1 1 o o : l Raffinose hendecaacetate 125.7 LRhamnitol pentaacetate 121’7 5OO:l (leYo)-Sorbitol hexaacetate 126.1 a Unpublished results of this Laboratory.

450 350 400 500 300 300 375 350

Lower Upper Lower Upper Lower Upper Lower Upper Lower Upper Lower Upper Lower Upper Lower Upper

--

Recovered materials Q ) D (20-25’ dmx, c < 5 j Accepted Yield Found value Wt., mg.

M. p., “C. Accepted Found value

129-130 159-160 130-131

86-88 129-130 99-100 130-131 64-66 158-189 8s-88 157-158 97-98 863-88 99-100 Sirup 97-98

132 160 132 89 132 100-101 132 70 160 89 160 100-101 89 100-101 Sirup 99

+ 4.0 +62 +41 +59 + 4.1 +99 + 3.9

+ 3.8 + 63 + 3.8 + 60

118.5 124.7 115.8 124.9 3 . 8 123.5 +101 122.5 3 . 8 115.0 35 113.8 +32 116.2 63 +62 119.4 60 +61 +63 63 122.0 122.9 +99 1-100 +61 60 123.4 122.2 +l00 +98 -31 - 30” 103.8 10 125.9 9.4

+

+

+ + + + + + +

% 95.5 95.7 92.4 99.4 97.6 98.8 91.7 89.2 92.8 94 3 97.9 98.0 98.6 97.4 85.3 99.7

THEPRESRNCE OF A PROTEOLYTIC ENZYME IN CASEIN

April, 1945

tives allows the zones to be visible without column extrusion, the p-phenylazobenzoates are not as well characterized as the acetates and are more difficult to prepare. Since the acetate group is relatively small, inherent differences in the carbohydrate structure are not so masked as when a larger substituent is employed. Thus, the sugar residue constitutes 44.9% of glucose pentaacetate but only 14.3% of glucose penta-pphenylazobenzoate.

Experimental The adsorbent employed was “Magnesol” (Westvaco Chlorine Products Co., South Charleston, West Virginia), a synthetic hydrated magnesium acid silicate (2Mg0. 5SiO,).” It is necessary to examine the extrusive and adsorptive properties of each new lot of this material. “Celite” (No. 535, Johns-Manville Co., New York, N. Y.) was used as a filter aid. A mixture of 5 parts (by wt.) of “Magnesol” and 1 part of “Celite” was extracted a t room temperature with a large volume of acetone and air-dried at room temperature. An amount of 50 g. of this mixture was packed in a chromatographic tube (35 mm. in diameter, 2-30 mm. in length) with the aid of suction. After the top of the column had been moistened with 10 cc. of benzene, there was added 5 cc. of a benzene solution containing ca. 2.50 mg. of a 1:1 mixture of the two sugar acetates to be separated. The amount and composition of the benzene (thiophene-free)-ethanol (absolute) solution then employed for developing each column is shown in Table 11. The column was extruded and immediately brushed with a freshly prepared 1% solution of potassium permanganate in 2.5 N sodium hydroxide. The zones were detected by the appearance in the brush streak of yellow to orange bands separated by green interzones of adequate width. The brush mark exhibited a series of color changes from an initial green, through a yellow and orange, to a final light brown. The green interzones were caused by the relatively slower reaction of the solvent on the column. Eventually, the brush streak assumed a uniform, light brown color.

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The average rates of appearance of the mnes are shown in Table I. The zones were cut out and the brush marks

removed with a scalpel. Elution of each zone was eflected with 126 cc. of acetone and the acetone was removed by evaporation a t room temperature. The properties and yields of the residues so obtained are shown in Table 11. Adequate purity was present without any further purification. In a typical separation, the chromatogram was developed in ninety minutes. The brush reagent located a 8-maltose octaacetate zone (36 mm. broad) 25 mm. below the top of the column and beneath this, separated by a 80-mm. interzone, the fl-D-glUCOSepentaacetate zone (22 mm. broad). Different lots of “Magnesol” may lead to variations in these data. Preliminary experiments indicated that the differences in adsorption affinity between the following pairs would be such that rechromatographing would probably be required for their separation: a- and 8-D-gIucose pentaacetate; a-D-arabinose tetraacetate and a-D-gIucose pentaacetate; a-D-arabinose tetraacetate and keto-& fructose pentaacetate; D-mannitol hexaacetate and (1euo)sorbitol hexaacetate; fl-D-glucose pentaacetate and (levo)-sorbitol hexaacetate.

Summary

(12) U.S. Patent 2,076,545(1937); C. A . , 81,4023 (1937); U.S. Patents 2,163,525,2,163,527. 2,163,528 (1939); C. A , , 88, 8049

The chromatographic brush method with aqueous alkaline permanganate as brush reagent and “Magnesol” as adsorbent has been employed successfully in the separation of a selected number of two component (equal portions) mixtures of fully acetylated sugars and sugar alcohols. The following separations were effected : 8-D-glucose pentaacetate (I) from &maltose octaacetate (11); I from sucrose octaacetate; I from keto-D-fructose pentaacetate ; I from raffinose hendecaacetate; I1 from sucrose octaacetate; I1 from raffinose hendecaacetate ; raffinose hendecaacetate from sucrose octaacetate; (levo)-sorbitol hexaacetate from L-rhamnitol pentaacetate. The components were recovered in nearly quantitative yield and in excellent purity.

(1939).

COLUMBUS, OHIO

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RECEIVED NOVEMBER 20, 1944

EASTERN REGIONAL RESEARCH LABORATORY,‘ PHILADELPHIA, PENNSYLVANIA]

.

On the Presence of a Proteolytic Enzyme in Casein BY ROBERTC. WARNERAN^ EDITHPOLIS

The presence of a proteolytic enzyme in milk was first reported by Babcock and Russell.2 They studied the decomposition of skim milk in the presence of antiseptics and the formation of soluble nitrogenous products during the ripening of cheese and attributed the observed proteolysis to an m y m e , “galactase.” Others**4have studied the same problem, and the general results of Babcock and Russell have been confirmed. No (1) One of the laboratories of the Bureau of Agricultural and Industrial Chemistry, Agricultural Research Administration, United States Department of Agriculture. Article not copyrighted. (2) Babcock and Russell, A i s . Rep., Wisconsin Agr. E*#.Sfo., 14, 161 (1897); Babcock,R u s l ~ l and l Vivian, ibid., IC, 77. 93 (1898). (3) Van Slyke. Harding and Hart, N. Y.Agr. Exp. Stn. Bulletin

loa, 21s (1901).

(4) Thatcher and M b e r g , J . Agr. &search, 11,487 (1917).

successful preparations of concentrates of the enzyme have been made. We have found that almost all of the proteolytic activity in milk is precipitated along with the casein when milk is acidified. The activity is extremely difficult to separate from casein and is absent from the usual casein preparations only if they have been exposed for long periods to alcohol. It is thus present in both commercial casein and most purified laboratory preparations. The fact that casein may undergo proteolysis in solution with the formation of products soluble a t PH 4.6 has been noted by Robertson6 and Walters.’ However, they believed that their (5) Robertson, J . Bid. Chem., 0 , 317 (1906-1907). (6) Walters. ibid., 11,267 (1912); ll, 43 (1912).